Neurotrophin antagonist compositions

ABSTRACT

A pharmaceutical composition comprising a compound of Formula I 
     
       
         
         
             
             
         
       
     
     wherein R 1  is selected from, inter alia, alkyl, aryl-loweralkyl, heterocycle-loweralkyl, loweralkyl-carbonate; optionally substituted amino; benzimidaz-2-yl; optionally substituted phenyl; loweralkyl-(R 5 )(R 6 ) wherein one of R 5  and R 6  is selected from H and the other is selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl; NHCH 2 CH 2 OX wherein X represents an in vivo hydrolyzable ester; and R 2  and R 3  are independently selected from H, NO 2 , halo, di(loweralkyl)amino, cyano, C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR 7  wherein Z is selected from C and S and R 7  is selected from H, loweralkylamino and arylamino; or pharmaceutically acceptable salts or certain in vivo hydrolyzable esters or amides thereof, in an amount effective to inhibit neurotrophin-mediated activity, and a suitable carrier, is described. 
     The compositions are useful to inhibit undesirable neurotrophin-mediated activity such as the neurite outgrowth that occurs in some neurodegenerative disease states.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 09/592,015, filedJun. 12, 2000, which is a continuation of U.S. Ser. No. 09/440,505,filed Nov. 15, 1999, which is a continuation of U.S. Ser. No.09/292,458, filed Apr. 15, 1999, which is a continuation of the U.S.designation of international application PCT/CA97/00779, filed Oct. 20,1997, which claims priority to GB9710904.5, filed May 27, 1997, andGB9621902.7, filed Oct. 21, 1996, the entire teachings of which areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to neurotrophin antagonists. Inparticular, the present invention relates to compositions comprising aneffective amount of a compound which inhibits or reduces undesirableneurotrophin activity, and a pharmaceutically acceptable carrier.

BACKGROUND OF THE INVENTION

A family of structurally and functionally related neurotrophic factorsexist which are collectively known as neurotrophins. The family ofneurotrophins includes the nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4(NT-4), neurotrophin-5 (NT-5) and neurotrophin-6 (NT-6).

The neurotrophins exhibit similar structural conformations, includingthree surface β-hairpin loops, a β-strand, an internal reverse turnregion, and N- and C-termini. With respect to sequence similarities, theneurotrophins share approximately 50% amino acid identity. Theneurotrophins are also functionally similar in that they each exhibitlow affinity binding to a receptor known as the “p75 nerve growth factorreceptor” or p75^(NGFR). Each neurotrophin also exhibits binding to areceptor of the tyrosine kinase (trk) family which is of higher affinitythan the binding to the p75 receptor. This interaction is believed to berelated to neuron survival, but is also involved with neurondifferentiation including process formation. The trkreceptor-neurotrophin interaction has been found to be more selectivethan neurotrophin interaction with the p75^(NGFR) receptor. Inparticular, NGF binds only a trk receptor known as the TrkA receptor,while BDNF, NT-4 and NT-5 exhibit exclusive binding to a TrkB receptor.NT-3 is less selective and, although it binds primarily with a TrkCreceptor, it also exhibits some binding to the TrkA and TrkB receptors(Ibanez et al., EMBO J. 1993, 12:2281).

The neurotrophins function primarily to promote survival of certainclasses of peripheral and central neurons both during development andfollowing neuronal damage. NGF, in particular, is involved with thedevelopment of neurons in the peripheral nervous system and supportsneuronal survival, as well as enhancing and maintaining thedifferentiated state of neurons. However, in some neurological diseasestates, the neurotrophins may also support inappropriate neuriteoutgrowth thereby facilitating the progression of a disease condition.For example, neurotrophins promote the undesirable sprouting ofhippocampal “mossy fibres”. Such inappropriate sprouting of mossy fibresis a common accompaniment of epilepsy in humans. It is also postulatedthat the pain experienced by patients suffering from some chronic painsyndromes may be associated with sprouting of sensory pain fibersresponsive to NGF in particular into the spinal cord. In otherpathological states, such as Alzheimer's disease, aberrant processgrowth, known as dystrophic neurite formation, is a strong correlate ofdisease severity.

Thus, although the neurotrophins are essential for the normaldevelopment and growth of neurons, they may be detrimental under certaincircumstances. In such instances, ligands capable of inhibiting orreducing selected neurotrophin-mediated activities would be desirabletherapeutically to treat neurodegenerative diseases and conditionsincluding neuropathic pain and to repair nervous system injury.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide compositions capableof inhibiting, or at least reducing, undesirable neurotrophin-mediatedactivity.

In an aspect of the present invention, a composition is provided whichcomprises a carrier and an effective amount of a compound of Formula I:

whereinR¹ is selected from alkyl; aryl-loweralkyl; heterocycle-loweralkyl;loweralkyl-carbonate; amino optionally monosubstituted or disubstitutedwith a substituent selected from loweralkyl, aryl and hydroxyloweralkyl;benzimidaz-2-yl;

wherein R⁴ is phenyl optionally monosubstituted or disubstituted with asubstituent selected from loweralkyl and halo; phenyl optionallymonosubstituted or disubstituted with a substituent selected from amino,loweralkoxy, hydroxy and loweralkyl; NHCH₂CH₂OX wherein X represents anin vivo hydrolyzable ester, and loweralkyl-(R⁵)(R⁶) wherein one of R⁵and R⁶ is selected from H and loweralkyl and the other is selected fromcarboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R² and R³ areindependently selected from H, NO₂, halo, di(loweralkyl)amino, cyano,C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR⁷ wherein Z is selected from Cand S and R⁷ is selected from H, loweralkylamino and arylamino;and pharmaceutically acceptable salts thereof.

In a further aspect of the present invention, there is provided a methodfor inhibiting a neurotrophin-mediated activity comprising the step ofexposing neurons to a composition as described above.

A further aspect of the present invention provides a method forinhibiting neurotrophin-mediated activity in a mammal comprising thestep of administering a composition as described above to said mammal.

These and other aspects of the present invention will be described ingreater detail hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION

The term “alkyl” as used herein means straight and branched chain alkylradicals containing from one to eight carbon atoms and includes methyl,ethyl, propyl, isopropyl, t-butyl, pentyl, hexyl, heptyl, octyl and thelike.

The term “loweralkyl” as used herein means straight and branched chainalkyl radicals containing from one to four carbon atoms and includesmethyl, ethyl, propyl, isopropyl tert-butyl and the like.

The term “alkoxy” as used herein means straight and branched chainalkoxy radicals containing from one to eight carbon atoms and includesmethoxy, ethoxy, tert-butoxy and the like.

The term “loweralkoxy” as used herein means straight and branched chainalkoxy radicals containing from one to four carbon atoms and includesmethoxy, ethoxy, tert-butoxy and the like.

The term “aryl” as used herein means a 5 or 6 membered aromatic orheteroaromatic ring containing 1, 2 or 3 heteroatoms independentlyselected from O, N and S, and includes phenyl, pyridyl, thienyl,furanyl, pyrrolo, imidazolo and the like.

The term “heterocycle” as used herein means a five or six membered,non-aromatic ring optionally containing one or more double bonds and oneor two heteroatoms selected from O, S, and N, and includes dihydropyran,tetrahydropyran, tetrahydrofuranyl, azacyclohexane, azacyclohexene,dihydrothiapyran, tetrahydrothiapyran, morpholino and the like.

The term “halo” as used herein means halide and includes fluoro, chloro,bromo and iodo.

As used herein, in vivo hydrolyzable esters or amides are those readilyhydrolyzable esters or amides of compounds of Formula I, which are knownand used in the pharmaceutical industry and include α-acyloxyalkyl andesters of C₃₋₂₀-fatty acids.

As it is used herein, the term “neurotrophin” refers to neurotrophicfactors that are structurally homologous to NGF, i.e. include threesurface β-hairpin loops, a β-strand, an internal reverse turn region,and N- and C-termini, and which promote at least one of neuron survivaland neuron differentiation, as determined using assays of conventionaldesign such as the in vitro assay exemplified herein and described byRiopelle et al. in Can. J. of Phys. and Pharm., 1982, 60:707. Mammaliannerve growth factor (NGF), brain-derived neurotrophic factor (BDNF),neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-5 (NT-5) andneurotrophin-6 (NT-6) are examples of neurotrophins.

“Neurotrophin-mediated activity” is a biological activity that isnormally promoted, either directly or indirectly, in the presence of aneurotrophin. Neurotrophin-mediated activities include, for example,neurotrophin binding to the p75^(NGFR) receptor or neurotrophin bindingto one of the trk receptors, neuron survival, neuron differentiationincluding neuron process formation and neurite outgrowth, andbiochemical changes such as enzyme induction. A biological activity thatis mediated by a particular neurotrophin, e.g. NGF, is referred toherein by reference to that neurotrophin, e.g. NGF-mediated activity. Todetermine the ability of a compound to inhibit a neurotrophin-mediatedactivity, conventional in vitro and in vivo assays can be used. Forexample, a receptor binding assay, such as the assay described herein inExample 1, can be used to assess the extent to which a compound inhibitsneurotrophin/receptor binding. Inhibition of neurite survival andoutgrowth can be determined using the in vitro assay described byRiopelle et al. in the Can. J. of Phys. and Pharm, 1982, 60:707,illustrated herein in Example 2.

The present invention relates to compositions comprising an effectiveamount of a compound of Formula I, or pharmaceutically acceptable saltsor in vivo hydrolyzable esters or amides thereof (hereinafter referredto as a compound of Formula I), which inhibits neurotrophin-mediatedactivity, and a pharmaceutically acceptable carrier.

In embodiments of the invention, compounds of Formula I include those inwhich R¹ is selected from allyl; aryl-loweralkyl;heterocycle-loweralkyl; loweralkyl-carbonate; amino optionallymonosubstituted or disubstituted with a substituent selected fromloweralkyl, aryl and hydroxyloweralkyl; benzimidaz-2-yl;

wherein R⁴ is phenyl optionally monosubstituted or disubstituted with asubstituent selected from loweralkyl and halo; phenyl optionallymonosubstituted or disubstituted with a substituent selected from amino,loweralkoxy, hydroxy and loweralkyl; NHCH₂CH₂OX wherein X represents anin vivo hydrolyzable ester, and loweralkyl-(R⁵)(R⁶) wherein one of R⁵and R⁶ is selected from H and loweralkyl and the other is selected fromcarboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R² and R³ areindependently selected from H, NO₂, halo, di(loweralkyl)amino, cyano,C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR⁷ wherein Z is selected from Cand S and R⁷ is selected from H, loweralkylamino and arylamino;and pharmaceutically acceptable salts thereof.

In another embodiment of the invention, compounds of Formula I includethose in which R¹ is selected from aryl-loweralkyl;heterocycle-loweralkyl; loweralkyl-carbonate; amino optionallymonosubstituted or disubstituted with a substituent selected fromloweralkyl and hydroxyloweralkyl; benzimidaz-2-yl; NHCH₂CH₂OX wherein Xrepresents an in vivo hydrolyzable ester; and loweralkyl-(R⁵)(R⁶)wherein one of R⁵ and R⁶ is selected from H and loweralkyl and the otheris selected from carboxy, carboxy-loweralkyl and loweralkoxycarbonyl;and R² and R³ are independently selected from H, NO₂,di(loweralkyl)amino, and phenyl-S—; and pharmaceutically acceptablesalts thereof.

In a further embodiment of the invention, compounds of Formula I includethose in which R¹ is selected from amino optionally monosubstituted ordisubstituted with a substituent selected from loweralkyl andhydroxyloweralkyl; NHCH₂CH₂OX wherein X represents an in vivohydrolyzable ester; and loweralkyl-(R⁵)(R⁶) wherein one of R⁵ and R⁶ isselected from H and loweralkyl and the other is selected from carboxy,carboxy-loweralkyl and loweralkoxycarbonyl; and R² and R³ areindependently selected from H and NO₂; and pharmaceutically acceptablesalts thereof.

In a specific embodiment of the invention, compounds of Formula Iinclude:

-   N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;-   N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic    acid;-   N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;-   N-Furfuryl-1,8-naphthalimide;    —(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide;-   N-(Pyrid-2-ylethyl)-1,8-naphthalimide;-   1,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro-benzo[i]isoquinoline;-   2-{2-(4-Methylphenylsulphonanido)phenyl}-6-(N,N-dimethylamino)    naphthalimide;-   1,3-Dioxo-2-{2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4-tetrahydro-benzo[i]isoquinoline;-   N-Octyl-5-nitronaphthalimide;-   5-Bromo-1,3-dioxo-N-methylpyrid-3-yl-1,2,3,4-tetrahydrobenzo-[i]isoquinoline;-   1,3-Dioxo-5-nitro-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro[i]isoquinoline;-   6-Nitro-2-(tetrahydrofuran-2-ylmethyl)naphthalimide;-   N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;-   Naphthalicacid-N-aminoimide;-   2-{2-(4-Methylbenzsulphonamido)-4,5-dichlorophenyl}naphthalimide;-   3-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene;-   1,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   6-Nitro-2-(pyrid-3-methyl)naphthalimide;-   3-Amino-7,4-bis(ethyl-1,3-dioxo)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   2-(Benzimidaz-2-yl)-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   2-(2-Aminophenyl)naphthalimide;-   1,3-Dioxo-2-{4,5-dimethyl-2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   3-Methyl-3-(1,3-dioxo-5-nitro(1H,3H)benz[de]isoquinolyl)butyric acid    methylester;-   1,3-Dioxo-N-methyltetrahydrofurfur-2-yl-5-nitro-1,2,3,4-tetrahydro-[i]isoquinoline;-   N-(4-Ethoxyphenyl)-5-nitronaphthalimide;-   6-Nitro-2-(furfuryl)naphthalimide;-   Ethyl-5-nitro-1,3-dioxo-1H-benz[de]isoquinoline-2-3H-acetate;-   Naphthalicacid-N,N′-diimide;-   2-(2-Hydroxyphenyl)naphthalimide;-   5-Amino-N-butylnaphthalimide;-   1,3-Dioxo-5-nitro-n-propylmorpholino-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   6-Nitro-2-(pyrid-2-ylethyl)naphthalimide;-   N-Methylnaphthalimide;-   N-(Pyrid-2-ylmethyl)naphthalimide;-   N-(3,5-Dimethylphenyl)-1,8-naphthalimide;-   6-Bromo-N-dimethylamino-1,3-dioxo-1,2,3,4-tetrahydrobenzo-[i]isoquinoline;-   N-(1,3-Dioxo-6-phenylmercapto-1,2,3,4-tetrahydrobenzo[i]isoquinoline)-aminoethanol;    and-   N-Anilino-1,8-naphthalimide.

In a preferred embodiment of the invention, compounds of Formula Iinclude:

-   N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;-   N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic    acid;-   N-Acetoxy-1,3-dioxo-1,2,3,1-tetrahydrobenzo[i]isoquinoline;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;-   N-Furfuryl-1,8-naphthalimide;-   6-(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide;-   N-(Pyrid-2-ylethyl)-1,8-naphthalimide;-   1,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro-benzo[i]isoquinoline;-   2-{2-(4-Methylphenylsulphonamido)phenyl}-6-(N,N-dimethylamino)    naphthalimide;-   1,3-Dioxo-2-{2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4-tetrahydro-benzo[i]isoquinoline;-   N-Octyl-5-nitronaphthalimide;-   5-Bromo-1,3-dioxo-N-methylpyrid-3-yl-1,2,3,4-tetrahydrobenzo-[i]isoquinoline;-   1,3-Dioxo-5-nitro-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro[i]isoquinoline;-   6-Nitro-2-(tetrahydrofuran-2-ylmethyl)naphthalimide;-   N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;-   Naphthalicacid-N-aminoimide;-   2-{2-(4-Methylbenzsulphonamido)-4,5-dichlorophenyl}naphthalimide;-   3-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene;-   1,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   6-Nitro-2-(pyrid-3-methyl)naphthalimide;-   3-Amino-7,4-bis(ethyl-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   2-(Benzimidaz-2-yl)-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;    and-   2-(2-Aminophenyl)naphthalimide.

In a more preferred embodiment of the invention, compounds of Formula Iinclude:

-   N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)dione}-2-aminoethanol;-   N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic    acid;-   N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;-   N-Furfuryl-1,8-naphthalimide;-   6-(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide;-   N-(Pyrid-2-ylethyl)-1,8-naphthalimide; and-   1,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro-benzo[i]isoquinoline.

In a most preferred embodiment of the invention, compounds of Formula Iinclude:

-   N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;-   N-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic    acid;-   N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline; and-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol.

Another embodiment of the invention includes an in vivo hydrolyzableester or amide of a compound selected from the group consisting of:

-   N-{5-Nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic    acid;-   N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;-   N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol;-   Naphthalicacid-N-aminoimide;-   3-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene;-   1,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   3-Amino-7,4-bis(ethyl-1,3-dioxo)-1,2,3,4-tetrahydrobenzo[i]isoquinoline;-   2-(2-Aminophenyl)naphthalimide; and-   2-(2-Hydroxyphenyl)naphthalimide.

The compounds of the present invention can be prepared by techniqueswell known in the art Compounds of formula I wherein R¹, R² and R³ areas defined above can be prepared by reacting a 1,8-naphthalic anhydrideof Formula A with a primary amine of Formula B in a suitable solventsuch as toluene, methanol, ethanol, propanol or acetone and attemperatures in the range of 0° C. to the boiling point of the solventused. Both reagent A and reagent B are commercially available or can beprepared using procedures known to one skilled ii the art.

Acid addition salts of the compounds of Formula I are most suitablyformed from pharmaceutically acceptable acids, and include for examplethose formed with inorganic acids e.g. hydrochloric, sulphuric orphosphoric acids and organic acids e.g. succinic, maleic, acetic orfumaric acid. Other non-pharmaceutically acceptable salts e.g. oxalatesmay be used for example in the isolation of the compound of Formula Ifor laboratory use, or for subsequent conversion to a pharmaceuticallyacceptable acid addition salt. Also included within the scope of theinvention are solvates and hydrates of the invention.

The conversion of a given compound salt to a desired compound salt isachieved by applying standard techniques, in which an aqueous solutionof the given salt is treated with a solution of base e.g. sodiumcarbonate or potassium hydroxide, to liberate the free base which isthen extracted into an appropriate solvent, such as ether. The free baseis then separated from the aqueous portion, dried, and treated with therequisite acid to give the desired salt.

In vivo hydrolyzable esters or amides of certain compounds of Formula Ican be formed by treating those compounds having a free hydroxy or aminofunctionality with the acid chloride of the desired ester in thepresence of a base in an inert solvent such as methylene chloride orchloroform. Suitable bases include triethylamine or pyridine.Conversely, compounds of Formula I having a free carboxy group may beesterified using standard conditions which may include activationfollowed by treatment with the desired alcohol in the presence of asuitable base.

The compositions of the present invention are useful to inhibit orreduce undesirable neurotrophin activity both in vitro and in vivo.Thus, in an aspect of the invention, a composition comprising aneffective amount of a compound of Formula I and a suitable carrier isprovided. By “suitable carrier” is meant a carrier which admixes withthe compound of Formula I to yield a composition suitable for theapplication for which it is to be used. By “effective amount” is meantan amount of the compound sufficient to inhibit an undesiredneurotrophin-mediated activity to a measurable extent, preferably byabout 20%, more preferably by about 40%, most preferably by about 50%,as determined using assays of conventional design such as thosedescribed herein in the specific examples.

The present composition has use as a media supplement to preventundesirable neurotrophin-mediated activity of neuron cells in vitro. Forexample, primary sensory neurons require NGF for survival in cellculture; however, NGF also influences neuron differentiation, notablyprocess formation and outgrowth, which are undesirable for the use ofprimary sensory neurons in cell culture. Thus, to preserve neuronsurvival in vitro while inhibiting cell differentiation, NGF is added tothe cell culture media along with the compound of Formula I. Foraddition to the cell culture, the compound is first combined with acarrier which will not adversely affect the growth of the cells inculture. Such carriers will include, for example, physiologicallyacceptable fluids such as water or any other fluid suitable for additionto the cell culture. Alternatively, the compound can be combined withmedia suitable for culturing neuronal cells prior to being added to thecell culture. To be effective to prevent neuron differentiation, theconcentration of the compound in the cell culture will be in the rangeof from about 1-500 μM, and preferably from about 1-100 μM. The optimalconcentration of compound for use in preventing neuron differentiationin cell culture will, of course, vary depending on the extent ofinhibition desired as well as the type of neuronal cells involved.

Compositions for in vivo administration, e.g. for treating neurologicalconditions such as epilepsy or Alzheimer's disease, or for treatingchronic pain, are also contemplated. Such compositions comprise atherapeutically effective amount of the compound of Formula I togetherwith a pharmaceutically acceptable carrier. In this context, the term“pharmaceutically acceptable” means acceptable for use in thepharmaceutical and veterinary arts, i.e. non-toxic and not adverselyaffecting the activity of the compound. The term “therapeuticallyeffective amount” means an amount of the compound sufficient to reduceundesirable neurotrophin-mediated activity, as determined using assaysof conventional design, in an inflicted individual without causingadverse effects.

Pharmaceutically acceptable carriers useful to prepare compositions forin viva administration include conventional carriers used in formulatingdrugs, such as diluents, excipients and the like. Reference may be madeto “Remington's Pharmaceutical Sciences”, 17th Ed., Mack PublishingCompany, Easton, Pa., 1985, for guidance on drug formulations generally.As will be appreciated, the pharmaceutical carriers used to preparecompositions in accordance with the present invention will depend on thedosage form to be used to treat the inflicted individual.

According to one embodiment of the invention, a compound of Formula I isformulated for administration by injection intraventricularly, and isaccordingly provided as an aqueous solution in sterile and pyrogen-freeform and optionally buffered or made isotonic. Thus, the compound may beadministered in distilled water or, more desirably, in saline or 5%dextrose solution. Water solubility of the compound of the invention maybe enhanced, if desired, by incorporating into the composition asolubility enhancer, such as acetyltrimethylammonium bromide orchloride. Lyoprotectants, such as mannitol, sucrose or lactose andbuffer systems, such as acetate, citrate and phosphate may also beincluded in the formulation, as may bulking agents such as serumalbumin.

For use in treating individuals with a neurological condition, precisedosage sizes of a pharmaceutical composition appropriate for treatmentare established in appropriately controlled trials, and will correspondto an amount of a compound of Formula I that reduces undesirableneurotrophin-mediated activity without causing intolerable side effectsto the individual being treated. It is anticipated that an effectivetreatment regimen for patients will involve the intraventricularadministration of dosages which achieve a level of the compound in thespinal fluid of the individual being treated of about 1-500 μM. It willbe appreciated, of course, that the dosage sizes required to attain thisin vivo concentration will vary according to the route ofadministration, the frequency of administration, on the individual beingtreated and on the neurological condition being treated.

Specific embodiments of the present invention are described in moredetail in the following examples which are not to be construed aslimiting.

EXAMPLE 1 Binding of [¹²⁵I]NGF to PC12 Cells in the Presence and Absenceof BDNF

The ability of the compounds of Formula I to antagonize NGF interactionwith the p75 and trkA receptors was determined as follows.

(A) Iodination of NGF

NGF was labelled using the Lactoperoxidase labelling method (Sutter etal., J. Biol. Chem., 1979) and the labelled NGF was separated fromradiolabelling agents and free iodide using a PD-10 Sephadex G-25column.

(B) Cell Culture and Cell Preparation

PC12 cells were grown in RPMI with 10% heat inactivated donor horseserum and 5% fetal calf serum. Cells were harvested for binding bywashing off the media with calcium-magnesium free balanced salt solution(Gey's solution) and incubated in 5 ml Gey's solution at 37° C. for 15minutes. Cells were pelleted by centrifugation and suspended inHepes-Krebs Ringer buffer (HKR) (10 mM Hepes pH7.35, containing 125 mMNaCl, 4.8 mM KCl, 1.3 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO₄. 1 mg/ml BSAand 1.0 mg/ml glucose) at a concentration of 4×10⁶/ml and kept on ice.

(C) NGF Binding

The reaction was performed in a 96 well plate. Suspended cells (150 ul,10⁶ cells) were added to ¹²⁵I-NGF (final concentration of 0.5 mM) andthe competing compound of Formula I in a final volume of 300 ul of HKRbuffer. The plates were incubated with shaking for 2 hr at 4° C. At theend of the incubation, 100 ul aliquots of the reaction sample were addedto 400 ul microcentrifuge tubes containing 200 ul of 10% glycerol in HKRbuffer. The tubes were centrifuged for 1 minute at ˜5000 rpm and the tipcontaining the cell pellet was cut off. Radioactivity bound to the cellswas determined by measuring the ¹²⁵I-NGF associated with each pellet ina gamma counter. Specific binding is calculated as the differencebetween the amount of ¹²⁵I-NGF bound in the absence (total) and presence(NSB) of 50 nM unlabeled NGF. TrkA binding is determined similarlyexcept 10 nM BDNF is added to all reactions. Table 1 summarizes thevalues obtained from this experiment for the inhibition of binding ofNGF to P75 and TrkA by compounds of Formula I.

TABLE 1 % Inhibition Compound at 100 uMN-Dimethylamino-1,3-dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]iso- 59quinolineN-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)acetic 58acid N-Acetoxy-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline 57N-(1,3-Dioxo-5-nitro-1,2,3,4-tetrahydrobenzo[i]isoquinoline)-aminoethanol44 N-Furfuryl-1,8-naphthalimide 366-(N,N-Dimethylamino)-2-(benzimidazol-2-yl)naphthalimide 36N-(Pyrid-2-ylethyl)-1,8-naphthalimide 351,3-Dioxo-6-phenylmercapto-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro- 33benzo[i]isoquinoline2-{2-(4-methylphenylsulphonamido)phenyl}-6-(N,N-dimethylamino)- 29naphthalimide1,3-Dioxo-2-{2-(4-methylphenylsulphonamido)phenyl}-1,2,3,4- 29tetrahydrobenzo[i]isoquinoline N-Octyl-5-nitronaphthalimide 295-Bromo-1,3-dioxo-N-methylpyrid-3-yl-1,2,3,4-tetrahydrobenzo[i]- 28isoquinoline1,3-Dioxo-5-nitro-N-(pyrid-2-ylethyl)-1,2,3,4-tetrahydro[i]isoquinoline28 6-Nitro-2-(tetrahydrofuran-2-ylmethyl)naphthalimide 28N-(1,3-Dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline)aminoethanol 27Naphthalicacid-N-aminoimide 272-[2-(4-Methylbenzsulphonamido)-4,5-dichlorophenyl]naphthalimide 263-Nitro-1,8-(N-propioncarboxylate)succinamidonapthalene 251,3-Dioxo-2-(2-aminophenyl)-1,2,3,4-tetrahydrobenzo[i]isoquinoline 246-Nitro-2-(pyrid-3-methyl)naphthalimide 233-Amino-7,4-bis(ethyl-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline23 2-(Benzimidaz-2-yl)-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]isoquinoline23 2-(2-Aminophenyl)naphthalimide 201,3-Dioxo-2-[4,5-dimethyl-2-(4-methylphenylsulphonamido)phenyl]- 191,2,3,4-tetrahydrobenzo[i]isoquinolineN-(4-Ethoxyphenyl)-5-nitronaphthalimide 181,3-Dioxo-N-methyltetrahydrofurfur-2-yl-5-nitro-1,2,3,4-tetrahydro- 18[i]isoquinoline3-Methyl-3-(1,3-dioxo-5-nitro(1H,3H)benz[de]isoquinolyl)butyric acid 17methylester 6-Nitro-2-(furfuryl)naphthalimide 17Ethyl-5-nitro-1,3-dioxo-1H-benz[de]isoquinoline-2-3H-acetate 16Naphthalicacid-N,N′-diimide 13 2-(2-Hydroxyphenyl)naphthalimide 135-Amino-N-butylnaphthalimide 131,3-Dioxo-5-nitro-n-propylmorpholino-1,2,3,4-tetrahydrobenzo[i]- 11isoquinoline 6-Nitro-2-(pyrid-2-ylethyl)naphthalimide 11N-Methylnaphthalimide 11 N-(Pyrid-2-ylmethyl)naphthalimide 10N-(3,5-Dimethylphenyl)-1,8-naphthalimide 96-Bromo-N-dimethylamino-1,3-dioxo-1,2,3,4-tetrahydrobenzo[i]- 5isoquinolineN-(1,3-Dioxo-6-phenylmercapto-1,2,3,4-tetrahydrobenzo[i]iso- 3quinoline)aminoethanol N-Anilino-1,8-naphthalimide 1

EXAMPLE 2 Inhibition of Neurite Outgrowth

The ability ofN-{5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol(Compound A) to inhibit neurite outgrowth was determined using thefollowing assay.

Eight-day chick embryo dorsal root ganglia (DRG) were freed of meningesand removed aseptically. The DRG were kept at 4° C. at all times.Ganglia from six embryos (40-50 per embryo) were washed in Ca²⁺- andMg²⁺-free Gey's balanced salt solution (Gibco) and exposed to 0.01%trypsin (Worthington) in the same solution for 10 nm u at 37° C. Ahalf-volume of phosphate-buffered Gey's balanced salt solution was addedfor a further 5 min at 37° C. and the reaction was then terminated withone-third volume of Ham's F12 medium (Gibco) containing 5% fetal calfserum (FCS, Gibco). The ganglia were then triturated using a 5 mLnarrow-tip pipette to a single cell suspension. Following filtrationthrough 37-mm nylon mesh (Small Parts Inc., Miami, Fla.) in a Milliporechamber to remove clumps, the cell suspension was washed through a500-ml FCS undercut (700×g for 5 min at 4° C.) and resuspended in 4 mLof Ham's F12 medium plus 5% FCS. The cell suspension was then preplatedon a 100-mm Flacon culture dish and incubated for 45-60 min at 37° C. ina 5% CO, humidified atmosphere. Cells enriched in neurons were decantedfor the bioassay, since non-neuronal cells of DRG preferentially stickto the culture substrate.

The inside wells of 96-well Falcon microculture plates were coated withpolylysine (0.1 mg/mL) (Sigma) for 4 h at 37° C. (the outside wells werefilled with distilled water to provide humidity) and, following a rinsewith tissue culture media, 100 mL of neuron-rich cell suspension wasadded to each well at 10⁵ cells/mL. Ninety (90) mL of NGF solution(prepared in tissue culture media) was then added to each well to afinal concentration of 0.25 ng/mL NGF per well. Ten (10) mL of testcompound solution, i.e. tissue culture media admixed with a compound ofFormula I, was then added to test wells in duplicate to yield wellscontaining compound concentrations ranging from 0 μM-100 μM. For controlassays, 10 mL of Ham's F12 medium was added to duplicate NGF-containingwells. The plates were covered and incubated in the dark for 24-30 hrs.at 37° C. in a 5% CO₂ humidified atmosphere.

The bioassays were read using a Leitz Diavert microscope with phaseoptics. To afford adequate optics, the meniscus effect of each well wasremoved by filling the well with a balanced salt solution until a flat,air-filled interface was achieved at the top of the well. At least 100neurons per well were counted, and the assay was scored as the ratio ofcells bearing neurites greater than one cell diameter to total viable(phase bright) cells. These results are summarized in Table 2.

TABLE 2 Group Ratio Std Dev SEM NGF Control 1.000 0.16 0.06  5 μMCompound A 1.1 0.42 0.15  50 μM Compound A 0.16 0.15 0.05 100 μMCompound A 0.016 0.02 0.01

EXAMPLE 3 Animal Models of Neuropathic Pain

For pain related to nerve injury, the compoundN-{5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol(Compound A) was tested in nerve-ligated rats for activity againsttactile allodynia, thermal hyperalgesia and in direct production ofthermal antinociception. The nerve-ligation model is commonly acceptedas representing aspects of neuropathic pain reported by humans. Shamoperated rats served as appropriate controls for the neuropathicexperiments.

Nerve Ligation Injury:

Nerve ligation injury was performed according to the method described byKim and Chung (Pain 50: 355-363, 1992). Rats were anesthetized withhalothane and the vertebrae over the L4 to S2 region were exposed. L5and L6 spinal nerves were exposed, carefully isolated, tightly ligatedwith 4-0 silk suture distal to the DRG. After ensuring homeostaticstability, the wounds were sutured, and the animals were allowed torecover in the cages. Sham-operated rats were prepared in an identicalfashion except that the L5/L6 nerve roots are not ligated.

Intrathecal Catheter Placement:

The test compounds were injected through indwelling i.th. catheters.While under anesthesia, PE-10 tubing (8 cm) was inserted through anincision made in the atlanto-occipitil membrane to the level of thelumbar enlargement of the rat and secured. Drug injections were made ina volume of 5 μl of 50% aqueous DMSO followed by a 9 μl saline flush.

Endpoints: (A) Evaluation of Tactile Allodynia:

Mechanical alloydynia was determined in the manner described by Chaplanet al. (J. Neurosci. Meth. 53:55-63, 1994). The paw withdrawal thresholdwas determined in response to probing with calibrated von Freyfilaments. The rats were kept in suspended cages with mesh floors andthe von Frey filaments were applied perpendicularly to the plantarsurface of the paw of the rat until it buckled slightly, and held for3-6 sec. A positive response was indicated by a sharp withdrawal of thepaw. The 50% paw withdrawal threshold was determined by thenon-parametric method of Dixon (Ann. rev. Pharmacol. Toxicol. 20:441-462, 1980).

(B) Evaluation of Thermal Hyperalgesia:

Thermal hyperalgesia was determined by focusing a radiant heat sourceonto the plantar surface of the affected paw of nerve-injured orsham-operated rats. Paw withdrawal latencies are determined by aphotodetection device which halts the stimulus and the timer after amaximum cut-off of 40 sec to prevent tissue damage. The withdrawallatency of sham-operated rats were compared to those of ligated rats tomeasure the degree of hyperalgesia.

(C) Evaluation of Acute Nociceptive Responses:

Acute nociception was determined by using the nociceptive warm watertail-flick reflex. This test was performed by placing the tail of thenerve-injured or sham-operated rats in a heated water bath maintained at55° C. The latency until tail withdrawal (rapid flick) from the bath wasdetermined and compared among treatments. A 15 second cut-off wasemployed to avoid tissue damage.

Compound A and morphine were tested in the nerve-ligated injury model ofneuropathic pain using 3 routes of administration: intraopertoneally(i.p.), intra-thecally (i.th.) and intracerebroventrically (i.c.v.). Thecompounds were evaluated for three endpoints: tactile allodynia, thermalhyperalgesia and acute nociception. Compound A is not active when giveni.c.v. The results for i.p and i.th. administration are shown in Table3.

TABLE 3 Summary of the A₅₀ doses with 95% Confidence Limits (C.L.) forCompound A and Morphine in L5/L6 Nerve Ligated and Sham-Operated Rats inModels of Tactile Allodynia, Acute Nociception and Thermal HyperalgesiaCompound A Morphine Treatment A₅₀ (95% C.L.) A₅₀ (95% C.L.)Allodynia-Ligated i.th 34.6 μg >100 μg i.p. 38 mg/kg 7.1 mg/kg TailFlick-Ligated i.th 78 μg 2.3 μg i.p. 27.8 mg/kg na* Tail Flick-Sham i.th34 μg 1.45 μg i.p. 25.7 mg/kg na Hot Plate-Ligated i.th 14.8 μg na i.p.18.6 mg/kg na Hot Plate-Sham i.th 48.1 μg na i.p. 34.9 mg/kg na *na =not available

EXAMPLE 4 Preparation OFN-{5-nitro-1H-benz[de]isoquinoline-1,3(2H)-dione}-2-aminoethanol(Compound A)

3-Nitro-1,8-naphthalic anhydride (1 eq) and 2-hydroxyethylhydrazine (1eq) are dissolved in toluene and heated to reflux. The reaction ismonitored by tlc and halted when all of the starting materials areconsumed. The solvent is removed under reduced pressure and the productpurified, if necessary, by recrystallization or silica gelchromatography.

Other compounds of Formula I can be prepared in an analogous manner, orare available commercially from Ryan Scientific Inc., Isle of Palms,S.C., U.S.

1. A pharmaceutical composition comprising a compound of Formula I,

wherein R¹ is selected from alkyl; aryl-loweralkyl;heterocycle-loweralkyl; loweralkyl-carbonate; amino optionallymonosubstituted or disubstituted with a substituent selected fromloweralkyl, aryl and hydroxyloweralkyl; benzimidaz-2-yl;

wherein R⁴ is phenyl optionally monosubstituted or disubstituted with asubstituent selected from loweralkyl and halo; phenyl optionallymonosubstituted or disubstituted with a substituent selected from amino,loweralkoxy, hydroxy and loweralkyl; NHCH₂CH₂OX wherein X represents anin vivo hydrolyzable ester, and loweralkyl-(R⁵)(R⁶) wherein one of R⁵and R⁶ is selected from H and loweralkyl and the other is selected fromcarboxy, carboxy-loweralkyl and loweralkoxycarbonyl; and R² and R³ areindependently selected from H, NO₂, halo, di(loweralkyl)amino, cyano,C(O)OH, phenyl-S—, loweralkyl, and Z(O)OR⁷ wherein Z is selected from Cand S and R⁷ is selected from H, loweralkylamino and arylamino; andpharmaceutically acceptable salts thereof, in an amount effective toinhibit neurotrophin-mediated activity, and a suitable carrier. 2.(canceled)
 3. (canceled)
 4. (canceled)
 5. (canceled)
 6. (canceled) 7.(canceled)
 8. (canceled)
 9. (canceled)
 10. (canceled)
 11. (canceled) 12.(canceled)
 13. (canceled)